Gel dna purification kit

Total RNA was isolated from Peyer’s patches using RNAiso and immediately mRNA was reverse transcribed into cDNA using an ALV RT Kit (Takara, China) following manufacturer’s instructions. PCR primers were designed according to the gene sequence of VCAM-1 (GenBank accession No 174484.1). The forward primer was 5′- GAA TTC TCC CAA ATC GAC ATA TTC CC -3′ and the reverse primer was 5′- CTC GAG TTA TTT CTC TTG AAC AGT TAA TT -3′. The forward and reverse primers contained EcoRI and XhoI restriction sites, respectively. PCR was performed with the following parameters: pre-denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 30 s and extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR products were analysed by electrophoresis on 1% agarose gels containing ethidium bromide. The gel band containing the amplified product was isolated, from which DNA was recovered and purified using a Gel DNA Purification Kit (Takara), then ligated to the pMD18-T vector (Takara). The recombinant plasmid named pMD-18T/ VCAM-1 was identified by double-enzyme digestion and nucleotide sequencing.